CN112684066A - Method for detecting mesylate genotoxic impurities in varlitinib mesylate by gas chromatography - Google Patents
Method for detecting mesylate genotoxic impurities in varlitinib mesylate by gas chromatography Download PDFInfo
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Abstract
The invention discloses a method for detecting mesylate genotoxic impurities in varenib mesylate by gas chromatography, wherein the anti-tumor drug mesylate genotoxic impurities of mesylate which may be contained in the varenib mesylate are generally detected by a liquid chromatography-mass spectrometry or gas chromatography-mass spectrometry method at present; according to the method, a sample is derivatized by a derivatization solution of sodium iodide and vitamin C, a gas chromatograph is used for detecting mesylate substances in the varenib mesylate, a standard addition method is used for calculating mesylate impurities, the interference of a sample can be effectively reduced, the method accuracy is superior to that of a common external standard method and a common internal standard method, and the result of the methodological verification proves that the method has good sensitivity, precision and accuracy, low detection cost and strong operability, and is superior to that of a conventional liquid or gas method.
Description
Technical Field
The invention relates to the field of drug analysis, in particular to a gas chromatography method for detecting mesylate genotoxic impurities possibly contained in an anti-tumor drug.
Background
Varenib mesylate is a tyrosine kinase (RTK) receptor inhibitor that inhibits the kinase activity of the Vascular Endothelial Growth Factor (VEGF) receptors VEGFR1(FLT1), VEGFR2(KDR) and VEGFR3(FLT4), and additionally inhibits other RTKs associated with the pro-angiogenic and tumorigenic pathways, including Fibroblast Growth Factor (FGF), receptors FGFR1, 2, 3 and 4, and Platelet Derived Growth Factor (PDGF) receptors PDGFR α, KIT and RET.
The structural formula of the varlitinib mesylate is as follows:
the methanesulfonic acid esters are impurities possibly generated in the production process of the methanesulfonic acid ranvatinib, the impurities have a genotoxicity warning structure, and the content of the methanesulfonic acid esters needs to be strictly controlled according to the ICH M7 guiding principle. The methanesulfonic acid esters are easy to hydrolyze in a water phase system, are thermally unstable, have low sensitivity in the detection process and are difficult to accurately quantify.
In the quality control of the mesylate, in the research of determining mesylate impurities in pefloxacin mesylate by a GC-MS method disclosed in the 6 th volume of 6 th month, 6.2017, 6.7, of Chinese antibiotic impurities, a GC-MS method for detecting the mesylate is reported in the prior literature. The method needs a gas mass spectrometer, has high detection cost and is not beneficial to laboratory popularization. In the existing literature drug identification 2013, 9, 3, volume 3, 17, that is, "determination of methanesulfonic acid alkyl esters in imatinib mesylate by using derivative gas chromatography", a method for determining methanesulfonic acid ester substances by using a gas phase method is reported. The method is a peak area external standard method, when a test sample is the methanesulfonic acid ranvatinib, the interference of the test sample in the derivatization process is large, the recovery rate is lower than 80%, the method is low in accuracy, and the methanesulfonic acid ester substances cannot be accurately controlled.
Varlitinib mesylate is a multitarget kinase inhibitor useful for the treatment of thyroid cancer, advanced renal cell carcinoma and liver cancer. The methanesulfonic acid esters possibly existing in the synthetic route of the product has a genotoxicity warning structure. The limit control of such impurities is required to be carried out according to the medication period of the product not more than 10 years, and calculated according to lifetime medication. The requirement of the ICH M7 guideline is that the maximum daily dosage of 24 mg/day is 62.5ppm by 1.5 mug/day. Therefore, in order to effectively control the quality of the sample, a method suitable for detecting the mesylate on a common gas chromatograph needs to be found.
Disclosure of Invention
In order to overcome the defects of the existing method, the invention provides a method for detecting mesylate genotoxic impurities in varenib mesylate by using a gas chromatography.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for detecting mesylate genotoxic impurities in varlitinib mesylate by gas chromatography comprises the following steps:
(1) weighing sodium iodide and vitamin C, adding water to dissolve and dilute to obtain a derivatization solution;
(2) weighing a test sample of the varenib mesylate, placing the test sample into a headspace bottle, adding a solvent to dissolve in water bath at 60-100 ℃, adding the derivatization solution obtained in the step (1), and performing derivatization reaction to obtain a test sample solution;
(3) preparing a reference substance solution and a sample adding standard solution;
(4) measuring the contents of the mesylate genotoxic impurities in the test solution and the standard limit solution of the test by adopting a headspace sample injection gas chromatography, and calculating the contents of the mesylate genotoxic impurities in the methanesulfonic acid ranvatinib by adopting a standard addition method;
the solvent is a mixed solution of acetonitrile and water, and the volume ratio of the acetonitrile to the water is 60: 40; the mesylate genotoxic impurities comprise methyl mesylate, ethyl mesylate and isopropyl mesylate; the derivatization reaction is to use a derivatization solution to derivatize target substances, namely methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate into methyl iodide, ethyl iodide and iodoisopropanol.
Further, the mass volume ratio of sodium iodide to water in the step (1) is 0.8-1.2 g/ml; the mass volume ratio of the vitamin C to the water is 0.0005-0.0015 g/ml.
Further, the volume ratio of the solvent to the derivatization solution in the step (2) is 1: 6-3: 1; the mass-volume ratio of the test sample of the varlitinib mesylate to the solvent is 20-60 mg/ml.
Further, the control solution in the step (3) comprises a control stock solution, a 50% control solution, a 100% control solution and a 150% control solution; the sample labeling solution comprises sample labeling 50% limit solution and sample
Adding a standard 100% limit solution and a standard 150% limit solution to a sample, and the preparation steps are as follows:
(1) preparation of a control stock solution: weighing methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate, and adding a solvent for quantitative dilution to obtain a reference stock solution, wherein the mass-to-volume ratio of the methyl methanesulfonate, the ethyl methanesulfonate and the isopropyl methanesulfonate to the reference stock solution is 25-500 [ mu ] g/mL;
(2) preparation of 50% control solution: transferring the reference substance stock solution, adding a solvent for quantitative dilution to obtain a 50% reference substance solution, wherein the mass-volume ratio of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate to the 50% reference substance solution is 2.5 μ g/mL;
(3) preparation of 100% control solution: transferring the reference substance stock solution, adding a solvent for quantitative dilution to obtain a 100% reference substance solution, wherein the mass-volume ratio of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate to the 100% reference substance solution is 5 mug/mL;
(4) preparation of 150% control solution: transferring the reference substance stock solution, adding a solvent for quantitative dilution to obtain a 150% reference substance solution, wherein the mass-volume ratio of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate to the 150% reference substance solution is 7.5 μ g/mL;
(5) preparing a sample adding standard 50% limiting solution: taking 0.2g of a test sample of the varlitinib mesylate, placing the test sample in a headspace bottle, adding 2.0ml of a 50% reference solution, dissolving in a water bath, adding 3.0ml of a derivatization solution, and sealing to obtain a sample standard-adding 50% limit solution;
(6) preparing a sample adding standard 100% limiting solution: taking 0.2g of a test sample of the varlitinib mesylate, placing the test sample in a headspace bottle, adding 2.0ml of 100% reference solution, dissolving in a water bath, adding 3.0ml of derivatization solution, and sealing to obtain a limit solution of the test sample with the standard of 100%;
(7) preparing a standard-added 150% limiting solution of a test sample: taking 0.2g of a test sample of the varlitinib mesylate, placing the test sample in a headspace bottle, adding 2.0ml of a 150% reference solution, dissolving the test sample in a water bath, adding 3.0ml of a derivatization solution, and sealing to obtain a 150% limit solution of the test sample.
The solvent in the steps (1) to (4) is a mixed solution of acetonitrile and water, and the volume ratio of the acetonitrile to the water is 60: 40; the water bath temperature in the steps (5) to (7) is 60-100 DEG C
Further, the calculation method of the content of the toxic impurities of the mesylate genes in the step (4) is a standard addition method;
further, the standard addition method comprises the following calculation steps: respectively taking a sample solution, a sample solution adding 50% limit solution, a sample adding 100% limit solution and a sample adding 150% limit solution for gas chromatography detection, recording a chromatogram, sequentially peaking iodomethane, iodoethane and iodoisopropane, and drawing a standard curve by taking the adding content (mu g/ml) of each component reference substance as an X axis and the peak area as a Y axis; calculating the content of each component in the test sample according to the following formula;
methyl or ethyl or isopropyl methanesulfonate (%) ═ a | × 10-3×5/200×100；
Wherein A is the intercept of the standard curve on the X axis; 200 is the sample size of the sample, mg.
Further, the sample injection method adopting the gas chromatography in the step (4) is a headspace sample injection method, and the determination conditions are as follows:
the headspace equilibrium temperature is 70-130 ℃;
the headspace balance time is 15-45 min;
the chromatographic column is a capillary chromatographic column;
the column length is 30m, the diameter is 0.32mm, and the film thickness is 1.8 μm;
column temperature: adopting temperature programming, wherein the initial column temperature is 35-45 ℃, and maintaining for 5 minutes; heating to 200 ℃ at a heating rate of 10-30 ℃/min for 10 minutes;
the temperature of a sample inlet is 180-220 ℃;
the temperature of a detection port is 210-250 ℃;
the column pressure is 40-60 KPa;
the flow rate is 2-5 ml/min;
the split ratio is 5: 1-20: 1;
the sample volume is 1 ml;
the gas chromatography detector is a hydrogen flame ionization detector.
Further, the capillary chromatography column is one of medium polarity, polar or a capillary column suitable for separating basic substances.
Further, the capillary chromatographic column is one of Aglient DB-WAX, Agilent CP-Volamine and Aglient DB-624; a preferred capillary chromatography column is the active DB-624.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the method adopts a standard addition method, can effectively deduct the detection interference caused by the test sample, obviously reduce the matrix interference of the main component, can accurately carry out quantitative analysis on the mesylate impurities, has accurate and reliable result, good sensitivity, precision and accuracy, and especially has obvious advantages in the aspect of accuracy;
(2) the method provided by the invention can be used for detecting the mesylate-based toxic impurities only by using the gas chromatography, has strong operability, reduces the detection cost and is convenient to popularize and apply in general laboratories.
Drawings
FIG. 1 is a gas chromatogram for methyl methanesulfonate (methyl iodide) location;
FIG. 2 is a gas chromatogram for the localization of ethyl methanesulfonate (iodoethane);
FIG. 3 is a gas chromatogram of isopropyl methanesulfonate (iodoisopropane) location;
FIG. 4 is a gas chromatogram of a test solution;
FIG. 5 is a gas chromatogram of a sample with a standard 50% limiting solution;
FIG. 6 is a gas chromatogram of a sample with a standard 100% limiting solution;
FIG. 7 is a gas chromatogram of a sample with a standard 150% limiting solution;
FIG. 8 Standard Curve for methyl methanesulfonate (methyl iodide);
FIG. 9 ethyl methanesulfonate (iodoethane) standard curve;
FIG. 10 is a standard curve of isopropyl methanesulfonate (iodoisopropane).
Detailed Description
The technical solutions provided by the present invention will be described in detail below with reference to specific examples, and it should be understood that the following specific embodiments are only illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1
1) Preparing a test solution:
(1) solvent: acetonitrile and water in a volume ratio of 60: 40, mixing;
(2) derivatization solution: weighing 20g of sodium iodide and 20mg of vitamin C, precisely weighing, adding water to dissolve and diluting to 20mL to obtain a derivatization solution containing about 1g of sodium iodide and about 1g of vitamin C1mg in each 1 mL.
(3) Blank solution: weighing 10g of sodium iodide and 10mg of vitamin C, precisely weighing, adding water to dissolve, and diluting to 10ml to obtain a blank solution containing 1g of sodium iodide and 1g of vitamin C1mg in each 1ml of solution.
(4) Control stock solutions: respectively weighing 25mg of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate, precisely weighing, and quantitatively diluting with 100mL of solvent to obtain solutions containing 250 μ g of each of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate in 1mL, as control stock solutions.
(5) 50% control solution: 1mL of the control stock solution was removed precisely, and diluted with 100mL of a solvent to prepare a 50% control solution containing about 2.5. mu.g each of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate per 1 mL.
(6) 100% control solution: 2mL of the control stock solution was removed precisely, and diluted with 100mL of a solvent to give solutions containing 5. mu.g each of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate per 1mL, as 100% control solutions.
(7) 150% control solution: 3mL of the control stock solution was removed precisely, and diluted with 100mL of a solvent to prepare a solution containing about 7.5. mu.g each of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate per 1mL of the solution as a 150% control solution.
(8) Test solution: taking about 0.2g of a test sample of the varlitinib mesylate, precisely weighing, placing in a headspace bottle, adding 2.0ml of a solvent, dissolving in a water bath at 80 ℃, adding 3.0ml of a derivatization solution, and sealing to obtain the test sample solution.
(9) Adding standard 50% limit solution to the test sample: taking about 0.2g of a test sample of the varlitinib mesylate, precisely weighing, placing the test sample in a headspace bottle, adding 2.0ml of a 50% reference solution, dissolving the test sample in a water bath at 80 ℃, adding 3.0ml of a derivatization solution, sealing, and adding a standard 100% limit solution as the test sample.
(10) Adding standard 100% limit solution to the test sample: taking about 0.2g of a test sample of the varlitinib mesylate, precisely weighing, placing in a headspace bottle, adding 2.0ml of 100% reference solution, dissolving in a water bath at 80 ℃, adding 3.0ml of derivatization solution, sealing, and taking the solution as a test sample added with a standard 100% limit solution.
(11) Adding standard 150% limit solution to the test sample: taking about 0.2g of a test sample of the varlitinib mesylate, precisely weighing, placing the test sample in a headspace bottle, adding 2.0ml of a 150% reference solution, dissolving the test sample in a water bath at 80 ℃, adding 3.0ml of a derivatization solution, and sealing to obtain a test sample added with a standard 150% limit solution.
(12) Preparing a positioning solution: methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate are respectively weighed, precisely weighed and placed in different measuring flasks, dissolved by adding a solvent and quantitatively diluted to a solution containing about 0.25mg of each component per 1ml, and the solution is used as a positioning stock solution; precisely measuring 1ml of each positioning stock solution, respectively placing in different headspace bottles, respectively adding 3ml of derivatization solution and 1ml of solvent, and sealing.
2) Specificity testing
The headspace sample introduction gas chromatography determination conditions are as follows:
the instrument comprises the following steps: agilent 7890B gas chromatograph; 7697A headspace auto sampler;
a detector: a hydrogen flame ionization detector;
a chromatographic column: an agent DB-624 capillary column, 30 mm by 0.32mm, 1.8 μm;
carrier gas: high-purity nitrogen;
sample inlet temperature: 200 ℃; detection of mouth temperature: 230 ℃;
column pressure: 50 KPa;
column temperature: adopting temperature programming: maintaining at 40 deg.C for 5min, raising the temperature to 200 deg.C at 20 deg.C/min, and maintaining for 10 min;
headspace conditions: headspace balance time 30 min; the headspace equilibrium temperature is 100 ℃;
the column flow rate was 3 mL/min;
the split ratio is 10: 1;
the sample volume is 1ml
The specificity determination method comprises the following steps: respectively taking the positioning solution, the test sample adding 100% limit solution and the blank solvent headspace sample injection, and recording the chromatogram.
The blank solvent was not interfered with the assay, and the retention time and the degree of separation of each compound are shown in the table below.
TABLE 1 analytical methods specificity results
In conclusion, under the condition of the chromatographic method, methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate components are respectively derivatized to generate methyl iodide, ethyl iodide and isopropyl iodide, and all the components are completely separated; no impurity peak in the solvent and the test solution interferes the detection of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate, and the method has good specificity.
Example 2 comparative validation by external standard method
Under the preparation item of the solution, the solvent, 100% of reference substance solution, sample standard adding 50% limit solution, sample standard adding 100% limit solution and sample standard adding 150% limit solution are injected in headspace under the chromatographic condition under the special item, and the recovery rate is determined by a peak area external standard method.
TABLE 2 results of recovery by external standard method
In conclusion, under the condition of the chromatographic method, methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate components are respectively derivatized to generate methyl iodide, ethyl iodide and isopropyl iodide, the recovery rate of each component is not in the range of 80-120%, and the accuracy of the method does not meet the requirement. It is necessary to improve the optimization method to eliminate the interference of the test sample.
Example 3 Standard addition verification
Respectively injecting sample solution, sample solution with standard 50% limit solution, sample solution with standard 100% limit solution, and sample solution with standard 150% limit solution in headspace, recording chromatogram, and calculating content of mesylate impurities according to standard addition method according to each chromatogram, as shown in FIGS. 4-7. In the chromatogram of the sample solution, iodomethane (methyl methanesulfonate), iodoethane (ethyl methanesulfonate) and iodoisopropane (isopropyl methanesulfonate) give peaks in sequence, and the separation degree between each component peak is greater than 1.5. And (3) taking the addition content (mu g/ml) of each mesylate control as an X axis and taking the peak area as a Y axis, and drawing a standard curve. The content of each mesylate in the test sample was calculated according to the following formula.
Methyl or ethyl or isopropyl methanesulfonate (%) ═ a | × 10-3×5/200×100
Wherein A is the intercept of the standard curve on the X axis; 200 is the sample size of the sample, mg.
TABLE 3 test results of Standard addition method
The standard addition method verifies that the corresponding standard curves of methyl methanesulfonate (methyl iodide), ethyl methanesulfonate (ethyl iodide) and isopropyl methanesulfonate (iodoisopropane) are shown in the attached figure 8, figure 9 and figure 10 of the specification.
And (4) conclusion: the standard addition method can deduct the interference of the test sample, the method has good linear relation, and the square of the linear correlation coefficient of the concentration addition amount of the methyl methanesulfonate, the ethyl methanesulfonate and the isopropyl methanesulfonate and the peak area is more than 0.995. Standard addition methods can be used for controlled detection of methane sulfonates in samples.
EXAMPLE 4 durability test
The other conditions were the same as in example 1 except that the headspace equilibrium time, headspace equilibrium temperature, injection port temperature, detector temperature, initial column temperature, temperature rise rate, column pressure, addition amount of sodium iodide in the derivatization solution, addition amount of vitamin C in the derivatization solution, and dissolution temperature of the test article were changed, respectively, to examine the durability of the method.
Respectively taking the sample solution prepared in the example 1, the sample standard-adding 50% limit solution, the sample standard-adding 100% limit solution and the sample standard-adding 150% limit solution, and measuring by headspace sample injection gas chromatography according to the parameters of tables 4-5; the results are shown in Table 6.
TABLE 4 durability test conditions (active DB-624, capillary column 30 m. times.0.32 mm, 1.8 μm)
TABLE 5 durability test conditions (different column types)
TABLE 6 results of methanesulfonic acid ester impurities detection under durability measurement conditions
The above results show that, by varying the chromatographic parameters, each of the durability conditions and the preferred conditions measures a difference in the content of each component of less than 20% of the limit (i.e. 62.5 x 0.2-17 ppm), and the maximum difference between methyl methanesulfonate and the preferred condition is 7.5 ppm; the maximum difference between ethyl methanesulfonate and the preferred conditions was 1.6 ppm; the maximum difference between isopropyl methanesulfonate and the preferred conditions was 1.2 ppm. Changing the type of the chromatographic column has no obvious influence on the separation and detection results of the methane sulfonate.
EXAMPLE 5 sensitivity test
The conditions for the headspace sample injection gas chromatography detection were the same as the preferred conditions in example 4.
A50% control solution (about 2.5. mu.g each of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate in 1 ml) was quantitatively diluted with a solvent to give a solution containing about 0.25. mu.g each of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate in 1ml, and the solution was used as a detection limiting solution.
A50% control solution (about 2.5. mu.g each of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate in 1 ml) was quantitatively diluted with a solvent to give a solution containing about 1. mu.g each of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate in 1ml, and the solution was used as a quantitative limiting stock solution.
Taking about 0.2g of a test sample of the varlitinib mesylate, precisely weighing, placing the test sample in a headspace bottle, adding 2.0ml of detection limit storage solution, dissolving in a water bath at 80 ℃, adding 3.0ml of derivatization solution, and sealing to obtain the detection limit solution.
Taking about 0.2g of a test sample of the varlitinib mesylate, precisely weighing, placing the test sample in a headspace bottle, adding 2.0ml of a quantitative limiting storage solution, dissolving the test sample in a water bath at the temperature of 80 ℃, adding 3.0ml of a derivatization solution, and sealing the solution to be used as the quantitative limiting solution.
Taking 5 parts of each quantitative limiting solution and 3 parts of each detection limiting solution, and sampling in headspace, wherein test results are shown in tables 7 to 9.
TABLE 7 results of detection limit and quantitation limit for methyl methanesulfonate
TABLE 8 detection limit and quantitation limit results for ethyl methanesulfonate
TABLE 9 results of detection limit and quantitation limit for isopropyl methanesulfonate
And (4) conclusion: the quantitative limit concentrations of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate are respectively 1.03. mu.g/ml, 1.03. mu.g/ml and 0.96. mu.g/ml, which are about 26ppm, 26ppm and 24ppm of the concentration of the test sample; the detection limit concentrations of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate are respectively 0.31. mu.g/ml, 0.31. mu.g/ml and 0.29. mu.g/ml, which are about 8ppm, 8ppm and 7ppm of the concentration of the test sample; namely, the contents of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate are respectively greater than 8ppm, 8ppm and 7ppm, and the contents are respectively greater than 26ppm, 26ppm and 24ppm, so that accurate quantification can be realized.
The technical means disclosed in the invention scheme are not limited to the technical means disclosed in the above embodiments, but also include the technical scheme formed by any combination of the above technical features. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and such improvements and modifications are also considered to be within the scope of the present invention.
Claims (10)
1. A method for detecting mesylate genotoxic impurities in varlitinib mesylate by using a gas chromatography method is characterized by comprising the following steps:
(1) weighing sodium iodide and vitamin C, adding water to dissolve and dilute to obtain a derivatization solution;
(2) weighing a test sample of the vareninib mesylate, placing the test sample into a headspace bottle, adding a solvent to dissolve in water bath at 60-100 ℃, adding the derivatization solution obtained in the step (1), and performing derivatization reaction to obtain a test sample solution;
(3) preparing a reference substance solution and a sample adding standard solution;
(4) measuring the contents of the mesylate genotoxic impurities in the test solution and the standard limit solution of the test by adopting a headspace sample injection gas chromatography, and calculating the contents of the mesylate genotoxic impurities in the methanesulfonic acid ranvatinib by adopting a standard addition method;
the solvent in the step (2) is a mixed solution of acetonitrile and water, and the volume ratio of the acetonitrile to the water is 60: 40;
the mesylate genotoxic impurities comprise methyl mesylate, ethyl mesylate and isopropyl mesylate.
2. The method for detecting the mesylate genotoxic impurities in the varlitinib mesylate according to claim 1, wherein the mass-to-volume ratio of sodium iodide to water in the step (1) is 0.8-1.2 g/mL; the mass-volume ratio of the vitamin C to the water is 0.0005-0.0015 g/mL.
3. The method for detecting the mesylate genotoxic impurities in the varlitinib mesylate by using the gas chromatography as claimed in claim 1, wherein the volume ratio of the solvent to the derivatization solution in the step (2) is 1: 6-3: 1; the mass-volume ratio of the test sample of the varlitinib mesylate to the solvent is 20-60 mg/ml.
4. The method for detecting the mesylate genotoxic impurities in the varlitinib mesylate by gas chromatography as claimed in claim 1, wherein the control solution in the step (3) comprises a control stock solution, a 50% control solution, a 100% control solution and a 150% control solution; the sample labeling solution comprises a sample labeling 50% limit solution, a sample labeling 100% limit solution and a sample labeling 150% limit solution, and the preparation steps are as follows:
(1) preparation of a control stock solution: weighing methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate, and adding a solvent for quantitative dilution to obtain a reference stock solution, wherein the mass-to-volume ratios of the methyl methanesulfonate, the ethyl methanesulfonate and the isopropyl methanesulfonate to the reference stock solution are respectively 25-500 [ mu ] g/mL;
(2) preparation of 50% control solution: transferring the reference substance stock solution, adding a solvent for quantitative dilution to obtain a 50% reference substance solution, wherein the mass-volume ratios of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate to the 50% reference substance solution are all 2.5 μ g/mL;
(3) preparation of 100% control solution: transferring the reference substance stock solution, adding a solvent for quantitative dilution to obtain a 100% reference substance solution, wherein the mass-volume ratios of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate to the 100% reference substance solution are respectively 5 mug/mL;
(4) preparation of 150% control solution: transferring the reference substance stock solution, adding a solvent for quantitative dilution to obtain a 150% reference substance solution, wherein the mass-volume ratios of methyl methanesulfonate, ethyl methanesulfonate and isopropyl methanesulfonate to the 150% reference substance solution are all 7.5 μ g/mL;
(5) preparing a sample adding standard 50% limiting solution: taking 0.2g of a test sample of the varlitinib mesylate, placing the test sample in a headspace bottle, adding 2.0ml of a 50% reference solution, dissolving in a water bath, adding 3.0ml of a derivatization solution, and sealing to obtain a sample standard-adding 50% limit solution;
(6) preparing a sample adding standard 100% limiting solution: taking 0.2g of a test sample of the varlitinib mesylate, placing the test sample in a headspace bottle, adding 2.0ml of 100% reference solution, dissolving in a water bath, adding 3.0ml of derivatization solution, and sealing to obtain a limit solution of the test sample with the standard of 100%;
(7) preparing a standard-added 150% limiting solution of a test sample: taking 0.2g of a test sample of the varlitinib mesylate, placing the test sample in a headspace bottle, adding 2.0ml of a 150% reference solution, dissolving the test sample in a water bath, adding 3.0ml of a derivatization solution, and sealing to obtain a 150% limit solution of the test sample.
5. The method for detecting the mesylate genotoxic impurities in the varlitinib mesylate according to claim 1, wherein the method for calculating the content of the mesylate genotoxic impurities in the step (4) is a standard addition method.
6. The method for detecting the mesylate genotoxic impurities in the varlitinib mesylate by using the gas chromatography as claimed in claim 1, wherein the sample injection method of the gas chromatography in the step (4) is a headspace sample injection method, and the determination conditions are as follows:
the headspace equilibrium temperature is 70-130 ℃;
the headspace balance time is 15-45 min;
the chromatographic column is a capillary chromatographic column;
the column length is 30m, the diameter is 0.32mm, and the film thickness is 1.8 μm;
column temperature: adopting temperature programming, wherein the initial column temperature is 35-45 ℃, and maintaining for 5 minutes; heating to 200 ℃ at a heating rate of 10-30 ℃/min for 10 minutes;
the temperature of a sample inlet is 180-220 ℃;
the temperature of a detection port is 210-250 ℃;
the column pressure is 40-60 KPa;
the flow rate is 2-5 mL/min;
the split ratio is 5: 1-20: 1;
the sample volume was 1 mL.
7. The method for detecting the mesylate genotoxic impurities in the varlitinib mesylate according to claim 4, wherein the solvent in the steps (1) to (4) is a mixed solution of acetonitrile and water, and the volume ratio of the acetonitrile to the water is 60: 40; the water bath temperature in the steps (5) to (7) is 60-100 ℃.
8. The method for detecting the mesylate genotoxic impurities in the varlitinib mesylate by using the gas chromatography method as claimed in claim 5, wherein the standard addition method comprises the following calculation steps: respectively taking a sample solution, a sample solution adding 50% limit solution, a sample adding 100% limit solution and a sample adding 150% limit solution for gas chromatography detection, recording a chromatogram, sequentially peaking iodomethane, iodoethane and iodoisopropane, and drawing a standard curve by taking the adding content (mu g/ml) of each component reference substance as an X axis and the peak area as a Y axis; calculating the content of each component in the test sample according to the following formula;
methyl methanesulfonate, ethyl methanesulfonate, or isopropyl methanesulfonate (%) = | A |. times.10-3×5/200×100；
Wherein A is the intercept of the standard curve on the X axis; 200 is the sample size of the sample, mg.
9. The method for detecting mesylate genotoxic impurities in varenib mesylate by gas chromatography as claimed in claim 6, wherein the capillary chromatography column is one of medium polarity, polar or capillary column suitable for separating basic substances.
10. The method for detecting the mesylate genotoxic impurities in the varenib mesylate by using the gas chromatography as claimed in claim 9, wherein the capillary chromatographic column is one of Aglient DB-WAX, Agilent CP-Volamine and Aglient DB-624.
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| CN114894939A (en) * | 2022-06-02 | 2022-08-12 | 江苏嘉逸医药有限公司 | Method for determining methanesulfonic acid alkyl ester in ambrisentan by gas chromatography |
| CN115684421A (en) * | 2022-11-04 | 2023-02-03 | 华夏生生药业(北京)有限公司 | Detection method and application of sulfonate impurities |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114894939A (en) * | 2022-06-02 | 2022-08-12 | 江苏嘉逸医药有限公司 | Method for determining methanesulfonic acid alkyl ester in ambrisentan by gas chromatography |
| CN115684421A (en) * | 2022-11-04 | 2023-02-03 | 华夏生生药业(北京)有限公司 | Detection method and application of sulfonate impurities |
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